Recent advances in the molecular genetics of deafness have vastly improved our ability to identify heritable hearing losses and have had several important consequences. First, the use of genetic testing to diagnose non-syndromic hearing loss (NSHL) has changed the medical evaluation of the deaf / hard-of-hearing person - after a history, physical examination and audiogram, genetic testing has become the next test ordered in the care of patients with presumed hereditary hearing impairment. Second, the identification of nearly one hundred genes that cause NSHL, coupled with technological advances in sequence capture and deep sequencing, has made robust epidemiological studies of genetic deafness possible for the first time. Third, understanding the genetic basis for normal auditory function is providing insight into inner ear physiology at the molecular level that will lad one day to the development of novel gene-specific and even mutation-specific therapies to treat hearing loss. Over the past granting period, we have focused on the following three specific aims: (1) To identify novel genetic causes of autosomal recessive non-syndromic hearing loss (ARNSHL); (2) To complete mutation screening of the coding sequence of all genes implicated in NSHL; (3) To study the Pendred syndrome / DFNB4 disease spectrum. In this competitive renewal, we will build on these accomplishments by completing the following specific aims: Specific Aim 1. To identify genetic mutations in regulatory elements that contribute to NSHL Hypothesis. Mutations in regulatory elements contribute to NSHL. These mutations can be identified by: a) studying a carefully selected patient population that is enriched for non-coding disease-causing variants and for whom full phenotypic and clinical data are available; and, b) applying targeted sequence capture and massively parallel sequencing to regulatory regions coupled with integrated analysis of murine ChIP-Seq data, ENCODE data and in silico data. Specific Aim 2. To compare simple and complex haplotypes in two well-defined populations - one with age-related hearing loss and one with normal hearing. Hypothesis. Custom modifications of the Galaxy pipeline make it possible to reconstruct unphased haplotypes for all NSHL genes included on OtoSCOPE(R). By studying two well-defined populations, one with and the other without age-related hearing loss, we will be able to identify at-risk and protective haplotypes and haplotype combinations that are associated with age-related hearing loss. The successful completion of these aims will have a major impact on our understanding of genetic deafness and offer novel insights into how specific protein-protein interactions contribute to age-related hearing loss.